Nowadays somatic embryogenesis is being investigated with special attention paid to the identification of genes directly involved in triggering of cell competence and development of ontogenic stages. In order to trace successive cell divisions that lead to the formation of the globular structures, modern scanning and electron microscopy methods are applied. Plantlets, which come from somatic embryogenesis process, should be exactly the same as mother plants but in vitro culture conditions may induce many disturbances, which could be lasting (hereditary) or only transitory. These changes are usually called somaclonal variation and could be observed on different levels of the plant organization. To investigate this kind of variations both of the genetic and epigenetic types, specially designed molecular systems are needed. Here, we describe induction of somatic embryos from several explants of G. kurroo. In order to evaluate the particular ontogenic stages of somatic embryos and variability of regenerated plants, following methods were applied: light microscopy, scanning and transmission electron microscopy, 2D protein electrophoresis, flow cytometry of DNA content in the cell nucleus, cytogenetic analysis of chromosome number and molecular analysis with the use of AFLP.
The possibility of induction of somatic embryogenesis in Polish and Chinese soybean cultivars was studied. Cotyledons from immature embryos as explants were used. Somatic embryos were obtained on MS medium with 40 mg l-1 of 2,4-D after 3-4 weeks of culture. Embryos appeared on 45,5% of Polan explants and the highest average number of somatic embryos per explant ? 29,2 was produced by cultivar Polan, too. The best development of somatic embryos was obserced on the MS medium with 0,5 mg l-1 KT and 0,5 mg l-1 BA and next on the hormone free medium with 60,0 g l-1 of sucrose and 0,5% activated charcoal.
Recently, several papers regarding genes essential for somatic embryogenesis have been published. The most important genes playing a key role in both zygotic and somatic embryogenesis are: SERK, LEC, and BBM. The majority of them are regulatory genes coding transcriptional factors. It was proved that the highest transcript accumulation of AGL15, BBM, SERK genes is characteristic for early stages of embryogenesis. The other genes, e.g. LEC1, L1L, LEC2, FUS3, PEI1 are preferentially expressed in later stages. Recently, NiR gene coding ferredoxin ? nitrite reductase, isolated from QTL region has been proved to play a key role in regeneration ability of rice. Although many investigations have been performed up to date, the molecular mechanisms of somatic embryogenesis is still far from understanding.
The transformation was led by pPR97/GUS/Intron LLpr10.1a promoter gene, which codes the acid protein. Two different media were used in the experiment. On Blayds medium, the highest efficiency of soamatic embryogenesis (25%) was observed in the Kometa variety. The culture media developed by Guelph University appeared to be the best for the Kama variety (18%).
Slices of scales and leaves isolated from Leucojum aestivum L. bulbs were cultured in the presence of auxins (NAA, 2,4-D, Picloram) and cytokinin, BA at different proportions and concentrations. The medium free of growth regulators was also used. Regeneration occurred as a result of somatic embryogenesis and organogenesis. Explants cultivated on the media containing 2,4-D and Picloram showed a capacity of somatic embryogenesis, viz. formation of embryogenic callus and somatic embryos. On the other hand, direct organogenesis (induction of bulblets and roots) occurred in explants grown on the medium devoid of growth regulators and enriched in NAA.
Modification of the medium composition was a trial to improve germination and conversion in somatic embryos of Norway spruce. The mature embryos germinated for 1 week in the medium supplemented with either auxin (IAA, IBA, NAA ? 0.05; 0.25; 0.1 or 0.5 muM) or gibberellin (GA3, GA4+7 - 25; 50; 125 or 250 mg dm-3). The hormone-free medium used for the next 2 weeks of germination was enriched or not with activated charcoal (10 mg dm-3). The auxin applied in the medium did not affect embryo germination nor root and shoot growth. GA3 stimulated root and shoot growth in germinating somatic embryos, whereas the highest concentration of GA4+7 completely inhibited embryo germination.
The aim of this study was to evaluate in vitro embryogenic potential of selected sweet potato [Ipomoea batatas L. (Lam.)] cultivars. Leaf blade and petiole from Ipomoea batatas plants (genotypes Rojo Blanco, White Jewel, White Star, Nemanete, Papota, and IIA-TIB 10) grown in pots and in vitro cultures were used for experiments as primary explants. The potential of embryogenic callus induction and somatic embryo development was evaluated for the cultures grown on the media supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BAP), and/or abscisic acid (ABA), under different light conditions (photoperiod, constant darkness). The cultivars appeared to differ with regards to embryogenic response of explants, somatic embryogenesis progression as well as embryo development and plant regeneration potential. The highest frequency of somatic embryogenesis was found for cv. Rojo Blanco and embryo development resulted in fully regenerated, healthy plants. In other cultivars, somatic embryo induction occurred, but embryogenesis has been arrested at the globular stage. White Star appeared to be the only completely recalcitrant cultivar which did not show any embryogenic response.
The aim of presented work was to investigate the impact of various light spectra on the efficiency and intensity of wheat somatic embryogenesis. The efficiency of somatic embryogenesis was defined as a percentage of explants forming somatic embryos in reference to all cultured explants. Immature embryos at the spherical coleoptile stage were excised from seeds of a few varieties of wheat and placed onto MS medium (1962) supplemented with 30 muM Dicamba. The influence of blue, white and red light on the callus growth and induction of somatic embryogenesis was compared. Increase in proportion between the red (600-700nm) and blue (400-500nm) component of light spectrum accelerated development of somatic embryos and increased the efficiency of somatic embryogenesis.
Ten Polish red clover cultivars were tested in vitro for their capability to produce callus and somatic embryos. A three-step tissue culture protocol (callus induction, embryo induction and plant development) based on Gamborg's B5 basal salts, NAA and 2,4-D as auxins, and kinetin and adenine as cytokinins was utilized. Explants source were petiole and hypocotyl sections. All clover varieties under study have showed high efficiency of callus production, and low ability for plant regeneration from the callus. The highest percent of genotypes which developed embryos was observed on variety Parka (3,6). Single embryogenic genotypes were selected from nearly all varieties. Hypocotyls showed the greatest ability for callus regeneration comparing to leaf petioles.
In this study we examined the effect of 2,4-D, NAA and picloram at 1.5, 3.0 and 4.5 mg/l in MS medium on callus induction and somatic embryogenesis in Asparagus densiflorus cv. Sprengeri and Asparagus plumosus. The callus formation of both shoot tips and nodal explants from A. densiflorus after 8 weeks of culture on MS medium supplemented with NAA and picloram (1.5, 3.0 mg/l) was observed. However, callus on medium containing 2,4-D was occasionally induced. On medium supplemented with picloram shoot tip explants of Asparagus plumosus showed callus inducing capacity. After four weeks of callus culture on hormone-free medium the globular embryos were achieved. On callus induced NAA organogenesis was observed.
We examined the toxicity of two antibiotics belonging to the betalactam group, to carrot (Daucus carota L.) protoplasts. Leaf protoplasts were cultured in the presence of cefotaxime or carbenicillin applied in five concentrations in the range from 0.1 to 0.5 mg ml-1. Cell viability, division frequency, and regeneration capacity were assessed to determine the potential toxic effect of the antibiotics. Both antibiotics significantly reduced protoplast viability and their ability to divisions. Their toxic effect intensified linearly with increasing antibiotic concentrations in the culture medium. More pronounced negative effect exhibited carbenicillin, which was evident 24 h after protoplast isolation. It also lowered cell mitotic activity two- to ten-fold, as compared to the control. Despite different reaction of cells exposed to carbenicillin and cefotaxime, callus tissue and somatic embryos were successfully obtained and allowed efficient plant regeneration. The comparison of the obtained results indicates that cefotaxime used in concentrations up to 0.2 mg ml-1 can be recommended in carrot cell cultures to prevent microbial contamination.
Based on the available literature, this article describes the advances made in cell culture of wheat. The importance of the age and physiological stage of the explant is discussed. The influence of the genotype is observed. The role of the components of the induction medium and in particular the role of auxin and kinetin, is investigated. The development of off-white callus and long-term culture of this callus facilitates the establishment of suspension culture - a source of totipotent protoplasts. This paper also focuses on the techniques which are used to introduce genes into wheat plants and on the somaclonal variation that occurs in a population of plants regenerated after tissue culture.
Somatic embryogenesis is a relatively new method of vegetative propagation of conifers. Many possibilities of its application include: cloning of trees for reforestration and timber production ? varieties resistant to environmental stresses, pests and diseases, as well as valuable ornamental varieties; conservation of rare and endangered species; provision of cell lines and protoplasts for genetic manipulations; germplasm preservation; synthetic seed production and use in basic research on conifer genetics and development. Conifer somatic embryogenesis was first reported in Picea abies (1,2) and Larix decidua, in 1985 (3). Since then this technology has been applied to more than 30 coniferous species from the genera: Abies, Larix, Picea, Pinus, Pseudotsuga, Sequoia, Taxus (4-7). As the general development pattern is similar for many conifers, the protocol of Norway spruce can be used as a model system of somatic embryogenesis in conifers. This protocol can be divided into six stages: 1) induction of embryogenic structures, 2) proliferation of embryogenic cultures, 3) maturation of somatic embryos, 4) embryo desiccation, 5) in vitro germination and conversion, and 6) transfer of emblings to soil (8).
In vitro cultures are an integral part of plant transformation. Genetic manipulation can be performed only on a single cell level. Therefore in vitro culture and regeneration of plants from a single cell are very important for successful transformation In vitro culture of rye is more difficult to conduct than of others cereals. Difficulties with in vitro regeneration of rye seems to be the main factor limiting the development of rye transformation systems. Genetic transformation process includes three main steps, single cell transformation, selection of transgenic cells and regeneration of plants from single cells. Efficiency of each of these steps can influence the result of the transformation process. Therefore optimisation of those steps is very important.Transformation has been performed using the microprojecticle method and scutellum of rye embryos as a target. Two constructs have been used in cotransformation, pDB1 containing a marker bar gene (phosphinotricine acetylotransferase) for Basta herbicide resistance and a repoter uidA gen (beta-glucuronidase) and pAwact-Sec containing 196 bp fragment of a sec-1 gene in anti-sense orientation. Using pAWact-Sec construct we tried to block the expression of the endogenous gene sec-1 coding omega-secalin which is storage protein of rye grain. We were able to regenerate transgenic rye plants containing all introduced genes. The efficiency of sec-1 expression blocking was analysed by SDS-PAGE method. Among 50 analysed kernels of T1 generation we found 5 with lower omega-secalin level. Complete blocking of omega-secalin was not observed.
In Arabidopsis thaliana, in vitro culture of immature zygotic embryos on medium supplemented with 2,4-D results in formation of somatic embryos via direct embrogenesis (DSE). The analysis of the nature of signals/stimuli involved in determination of embryogenic response in cultured explants can reveal genetic and physiological mechanisms involved in plant embryogenesis. The key factors for DSE induction in A. thaliana are the developmental stage of the explant and the presence of 2,4-D in induction medium. The study was undertaken to analyze DSE efficiency under modified tissue culture conditions. The studied factors included: pH of induction medium, temperature during embryogenesis induction, polyamines and their precursors, genotype and origin of the explants (seed-grown or in vitro-regenerated donor plants). The significant increase of the DSE efficiency was indicated on media with higher pH (7.0-8.0) and in culture of the explants obtained from plants regenerated via secondary embryogenesis. Moreover, embryogenic potential of the regenerant-derived explants was also observed on medium lacking of 2, 4?D. Spermidine and precursors of polyamines (ornithine and arginine) included in induction medium as well as any of the tested temperatures (5,28,32C) did not stimulate the DSE efficiency in comparison to standard conditions. All 16 tested ecotypes displayed the ability for the DSE under standard culture conditions. DSE efficiency varied between the studied ecotypes, however, most of the genotypes (75%) showed high (60-100%) frequency of explants producing somatic embryos.
The effect of salicylic acid (SA) applied during the proliferation phase of cell suspension in vitro regeneration system of Medicago sativa L. on cell suspension growth, production of somatic embryos and their following development, germination and conversion were examined. SA is a potent inhibitor of the above processes. It is suggested that endogenous jasmonates are of some importance in regulation of somatic embryogenesis.
Somatic embryogenesis, resembling zygotic embryogenesis in vivo, is considered to be an efficient method of in vitro propagation of a number of agronomically important plant species including Medicago sativa L. and it offers an in vitro experimental system for studying the embryo development. Artificial seed technology is one of the important applications of the process. Induction of embryogenesis, embryo development and induction desiccation tolerance are affected by plant growth regulators. The review will focus on the effect of plant growth regulators: auxins, cytokinins, gibberellins, abscisic acid, jasmonates, ethylene and inhibitors on the synthesis and action of different phases of somatic embryogenesis in Medicago sativa L.
This review paper covers the results of tissue culture and biotechnology of rye (Secale cereale L.) published between 1990 and 2000. The following subjects were raised: somatic embryogenesis, haploid production, intergenetic hybridisation and transformation. Upon conclusion of all the results published to date, we may say that the progress of biotechnology in the case of rye is very limited in comparison to other cereals. However, a lot of work and efforts were involved to obtain the aforementioned result. Taking into account the progress in plant genetics and molecular biology, the authors deeply believe that the New Millennium will bring a brake-through in rye biotechnology.
coconut water. Somatic embryos were formed on the surface of cotyledons of immature embryos. The process of was observed only on cotyledons of whole explants. Unfortunately, cotyledons isolated from the embryo axis did not produce somatic embryos. In the subepidermal cell layer, the initial cell divisions leading to somatic embryo formation were observed. The number of regenerated somatic embryos varied from one to many per cotyledon. Somatic embryo derived plantlets formed very good root system in perlite supplemented with medium. Cytological and anatomical evidences of this process are presented in this paper.
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